Desensitization to pharmacological agents is an important consideration in the treatment of human diseases. The molecular events associated with desensitization include modification of receptors for signalling molecules. In the superfamily of G-protein coupled receptors (GPRs), desensitization is believed to involve phosphorylation of the receptors which in turn leads to receptor/G-protein uncoupling. The proposed experiments will critically test the hypothesis that a family of enzymes known as G-protein coupled receptor kinases (GRKs) phosphorylate and desensitize the GPRs. The functionally distinct m2 and m3 muscarinic cholinergic receptors (mAChR) will be used as models of GPRs that couple to attenuation of adenylyl cyclase and stimulation of phosphoinositide hydrolysis. Both the m2 and m3 receptors desensitize in response to agonist stimulation. Agonists have been shown to induce phosphorylation of m2 and m3 mAChR in intact cells, and this may correlate with desensitization. Experiments will be designed to determine if the functionally distinct receptors desensitize via similar or different mechanisms. The first specific aim is to define the role of identified "receptor-specific" protein kinases in the phosphorylation of the m2 and m3 mAChR using intact cell and in vitro approaches. Experiments will focus on the phosphorylation and regulation of the m2 and m3 mAChR by the isoforms of the "beta-adrenergic receptor kinases" (betaARKs) and newly identified GRKs to determine if they are "mAChR kinases". Experiments will be performed with purified mAChR and kinases in vitro and with cultured cells transfected with m2 or m3 cDNAs. The efficacy of the m2 and m3 mAChR as substrates will be compared to the betaAR. Knock out approaches will be used to eliminate isoforms of GRKs from cells to determine if these enzymes regulate the mAChR under physiological conditions. In addition, mAChR mutants will be prepared and tested for their abilities to undergo phosphorylation and desensitization. Chimeric m2/m3 mAChR will be used to define the mechanisms underlying receptor phosphorylation. The second aim is to identify and characterize potentially novel mAChR kinases in tissues where the mAChR are naturally expressed and known to undergo desensitization. Experiments will use purified and reconstituted m2 mAChR as substrates to detect kinases in extracts of mammalian heart. The third aim will be to determine the functional consequences of the phosphorylation mediated by the receptor-specific kinases. Effects of phosphorylation on receptor signalling and receptor/G-protein interactions will be defined. The roles of arrestins in the desensitization of the m2 and m3 mAChR will be determined. Taken together the proposed experiments should provide detailed insights into the mechanisms of phosphorylation and desensitization of the two model GPRs.